Cloning, and expression in Escherichia coli K-12, of the chromosomal hemolysin (phospholipase C) determinant of Pseudomonas aeruginosa

K. Coleman, G. Dougan, J. P. Arbuthnott

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A hemolysin determinant was cloned from P. aeruginosa PA103 by inserting Sau3a-generated DNA fragments between the BamHI sites of the λ replacement replacement vector WL47.1. A 9.5-kilobase HindIII fragment encoding the hemolysin was subcloned from this phage and inserted into the plasmid vector pHC79 to generate the recombinant plasmid pKC95. Escherichia coli K-12 strains harboring pKC95 exhibited zones of hemolysis after several days of growth on blood agar plates. Hemolysis was shown to be due to phospholipase C activity by using the chromogenic substrate p-nitrophenylphosphorylcholine. Deletion mutants of pKC95 were isolated, and polypeptides expressed from these plasmids were examined by using the E. coli minicell system. A polypeptide of 78,000 daltons was associated with phospholipase C activity. The hemolytic activity was cell associated when expressed in E. coli.

Original languageEnglish
Pages (from-to)909-915
Number of pages7
JournalJournal of Bacteriology
Issue number2
Publication statusPublished - 1 Jan 1983
Externally publishedYes

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